Fluorescence lifetime imaging by time‐correlated single‐photon counting

Abstract We present a time‐correlated single photon counting (TCPSC) technique that allows time‐resolved multi‐wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four‐dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x‐y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near‐perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two‐photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP‐YFP FRET. Microsc. Res. Tech. 63:58–66, 2004. © 2003 Wiley‐Liss, Inc.